The enzyme ribonucleotide reductase catalyzes the reduction of ribonucleoside diphosphates to deoxyribonucleoside diphosphates, and provides the precursors for NDA synthesis. Ribonucleotide reductase activity is under complex allosteric feedback control. Evidence from my laboratory and others on the mechanisms of T-lymphocyte toxicity associated with adenosine deaminase and purine nucleoside phosphorylase deficiencies suggests that deoxyribonucleoside triphosphate synthesis is crucial for the control of DNA synthesis. I plan to study the relationship of purine and pyrimidine metabolic events to DNA synthesis by biochemical genetic techniques in mammalian cell lines. Using well characterized stable mutants of the S49 mouse T-lymphoma line, I will determine whether aberrations in enzymes involved in deoxyribonucleotide synthesis alter the deoxyribonucleoside triphosphate (dNTP) pools during various stages in the cell cycle of synchronized proliferating cells. Currently available cell lines with either mutant ribonucleotide reductase, adenylosuccinate synthetase, or deoxycytidylate deaminase activities will be studied. The quantity, intracellular location, and activity of ribonucleotide reductase will be evaluated at different stages of the cell cycle. The last aim of this proposal involves the isolation and characterization of DNA polymerase Alpha mutants with either altered affinity for deoxyribonucleoside triphosphates, or an increase in the level of DNA polymerase Alpha activity. Aphidicolin and other drugs discussed in the proposal will be used for this selection. These experiments will help clarify whether dNTP pools are modulated by the allosteric regulation of ribonucleotide reductase during the cell cycle or whether additional mechanisms are operating. They will provide a clearer understanding of the toxicity of deoxyadenosine (a toxic metabolite in adenosine deaminase deficiency), deoxyguanosine (a toxic metabolite in purine nucleoside phosphorylase deficiency), thymidine, hydroxyurea, and other agents that affect ribonucleotide reductase activity and DNA synthesis.